This application targets key issues of high clinical significance. Building upon our model implicating systemic monocyte activation and trafficking in initiating the development of HIV neurological disease, we seek to determine (1) the extent and nature of HIV protease and reverse transcriptase mutations within the brain parenchyma that develop in response to highly active antiretroviral therapy (HAART), as well as the mechanisms whereby they arise, (2) the sources of HIV in cerebrospinal fluid (CSF), including the sources of drug-sensitive rebound virus and (3) the likelihood that the brain can seed the periphery with drug-resistant or-sensitive virus. To address these issues, we propose to molecularly clone and sequence a 1.2kb region of the HIV genome from post- mortem bone marrow and lymph node tissues, along with multiple regions of brain, including meninges and choroid plexus, collected from patients treated with multiple antiretrovirals. This region includes the entire protease gene and that portion of the reverse transcriptase (RT) gene, which harbors most of the known RT resistance mutations. Sequences recovered from RNA (cDNA) from the same specimens will also be examined and used to (1) address issues of HIV latency, particular latency in macrophages, and (2) identify site of ongoing viral replication. Towards determining temporal relationships between the HIV species in CSF and those in circulating leukocytes, we will also clone and sequence the protease-RT genomic fragment, as well as the V1-V4 region of the HIV envelope gene, from CSF, plasma and separated populations of blood monocytes and T-cells collected longitudinally from patients with and without neurological disease, receiving HAART. When available, ante-mortem and post-mortem specimens from the same patient will be examined. Along with identification of patterns of drug resistance mutations, phylogenetic analyses will include identification of evolutionary relationships among the intra-patient HIV quasispecies. Given our multi-organ approach with a focus on HIV in macrophages, the results of these studies are likely to provide new insight into the critical events leading to HIV disease progression, as well as virologic failure, including failure in the CNS, in the absence of antiretroviral resistance. Optimal treatment planning requires an accurate understanding of the mechanisms underlying these events.